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1.
PLoS One ; 15(7): e0236372, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706797

RESUMO

Enzymatic assays based on bacterial 3α-hydroxysteroid dehydrogenase are the method of choice for quantification of total bile acids (BAs) in serum. Although non-specific, it is generally considered precise and robust. The aim of this study was to investigate how changes in the BA spectrum might affect the reliability of the method. We measured standard solutions of twenty-three human and murine BAs using a commercial enzymatic assay and compared the measured vs. expected concentrations. Additionally, total BA concentrations in rat and human cholestatic samples with an abnormal BA spectrum were measured using an enzymatic assay, and a more specific LC-MS/MS method. We observed a great variability in the response of individual BAs in the enzymatic assay. Relative signal intensities ranged from 100% in glycocholic acid (reference) to only 20% in α-muricholic acid. The enzymatic assay markedly underestimated the BA concentrations in both human and rat cholestatic sera when compared to the LC-MS/MS assay. Our study indicated that the performance of an enzymatic assay largely depends on the BA spectrum, and the total concentration of BAs can be markedly underestimated. Samples with an atypical BA spectrum (viz. in rodents) should preferably be measured by other methods.


Assuntos
Ácidos e Sais Biliares/sangue , Ensaios Enzimáticos/métodos , 3-alfa-Hidroxiesteroide Desidrogenase (B-Específica)/química , Animais , Colestase/metabolismo , Humanos , Ratos , Reprodutibilidade dos Testes
2.
Nat Commun ; 11(1): 2971, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532990

RESUMO

APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy.


Assuntos
Citidina Desaminase/genética , Regulação Neoplásica da Expressão Gênica , Mutação , Neoplasias/genética , Proteínas/genética , Edição de RNA , Linhagem Celular , Linhagem Celular Tumoral , Citidina Desaminase/metabolismo , Ensaios Enzimáticos/métodos , Células HEK293 , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas/metabolismo , Interferência de RNA , Sequenciamento Completo do Exoma/métodos
3.
J Vis Exp ; (159)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32478756

RESUMO

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most γ-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.


Assuntos
Aciltransferases/metabolismo , Química Click/métodos , Ensaios Enzimáticos/métodos , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Animais , Ácidos Graxos , Fibroblastos/metabolismo , Fluorescência , Humanos , Lipoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteobactérias/metabolismo , Especificidade por Substrato
4.
PLoS One ; 15(4): e0231115, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32255808

RESUMO

Leber congenital amaurosis (LCA) is a group of severe congenital retinal diseases. Variants in the guanylate cyclase 2D gene (GUCY2D), which encodes guanylate cyclase 1 (ROS-GC1), are associated with LCA1 and account for 6%-21% of all LCA cases. In this study, one family with LCA1 was recruited from China. A combination of next generation sequencing and Sanger sequencing was used to screen for disease-causing mutations. We found three novel mutations (c.139delC, p.Ala49Profs*36; c.835G>A, p.Asp279Asn and c.2783G>A, p.Gly928Glu) in the GUCY2D gene. Proband III-2 carries mutations c.139delC and c.2783G>A, which are inherited from the heterozygous mutation carriers, II-2 (c.139delC) and II-3 (c.2783G>A) that possess c.139delC and c.2783G>A. Additionally, II-8 carries heterozygous mutation c.835G>A. Sanger sequencing was used to confirm the presence of the three novel mutations in other family members. Mutation c.139delC results in a truncated protein. Mutations c.835G>A and c.2783G>A significantly reduce the catalytic activity of ROS-GC1. Our findings highlight the gene variants range of LCA. Moreover, HPLC-coupled tandem mass spectrometry (HPLC-MS/MS) was used to analyze the concentration of 3',5'-cyclic guanosine monophosphate (cGMP), suggesting that HPLC-MS/MS is an effective alternative method to evaluate the catalytic activity of wild-type and mutant ROS-GC1.


Assuntos
GMP Cíclico/análise , Guanilato Ciclase/genética , Amaurose Congênita de Leber/genética , Receptores de Superfície Celular/genética , Membrana Celular/metabolismo , Pré-Escolar , China , Cromatografia Líquida de Alta Pressão , GMP Cíclico/metabolismo , Análise Mutacional de DNA , Ensaios Enzimáticos/métodos , Feminino , Guanilato Ciclase/metabolismo , Células HeLa , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Amaurose Congênita de Leber/diagnóstico , Masculino , Mutagênese Sítio-Dirigida , Mutação , Linhagem , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem
5.
Artigo em Inglês | MEDLINE | ID: mdl-32243770

RESUMO

The primary function of the arterial microvasculature is to ensure that regional perfusion of blood flow is matched to the needs of the tissue bed. This critical physiological mechanism is tightly controlled and regulated by a variety of vasoactive compounds that are generated and released from the vascular endothelium. Although these substances are required for modulating vascular tone, they also influence the surrounding tissue and have an overall effect on vascular, as well as parenchymal, homeostasis. Bioactive lipids, fatty acid derivatives that exert their effects through signaling pathways, are included in the list of vasoactive compounds that modulate the microvasculature. Although lipids were identified as important vascular messengers over three decades ago, their specific role within the microvascular system is not well defined. Thorough understanding of these pathways and their regulation is not only essential to gain insight into their role in cardiovascular disease but is also important for preventing vascular dysfunction following cancer treatment, a rapidly growing problem in medical oncology. The purpose of this review is to discuss how biologically active lipids, specifically prostanoids, epoxyeicosatrienoic acids, sphingolipids, and lysophospholipids, contribute to vascular function and signaling within the endothelium. Methods for quantifying lipids will be briefly discussed, followed by an overview of the various lipid families. The cross talk in signaling between classes of lipids will be discussed in the context of vascular disease. Finally, the potential clinical implications of these lipid families will be highlighted.


Assuntos
Ácidos Graxos/metabolismo , Microvasos/metabolismo , Fosfolipídeos/metabolismo , Esfingolipídeos/metabolismo , Animais , Ensaios Enzimáticos/métodos , Fluorometria/métodos , Humanos , Espectrometria de Massas/métodos , Transdução de Sinais
6.
Int J Mol Sci ; 21(1)2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31947885

RESUMO

Successful directed evolution examples span a broad range of improved enzyme properties. Nevertheless, the most challenging step for each single directed evolution approach is an efficient identification of improved variants from a large genetic library. Thus, the development and choice of a proper high-throughput screening is a central key for the optimization of enzymes. The detection of low enzymatic activities is especially complicated when they lead to products that are present in the metabolism of the utilized genetic host. Coupled enzymatic assays based on colorimetric products have enabled the optimization of many of such enzymes, but are susceptible to problems when applied on cell extract samples. The purpose of this study was the development of a high-throughput screening for D-glycerate dehydratase activity in cell lysates. With the aid of an automated liquid handling system, we developed a high-throughput assay that relied on a pre-treatment step of cell extract prior to performing the enzymatic and assay reactions. We could successfully apply our method, which should also be transferable to other cell extract-based peroxidase assays, to identify an improved enzyme for the dehydration of D-glycerate.


Assuntos
Proteínas de Bactérias/metabolismo , Ensaios Enzimáticos , Ácidos Glicéricos/metabolismo , Hidroliases/metabolismo , Engenharia de Proteínas , Sulfolobus solfataricus/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Evolução Molecular Direcionada/métodos , Ensaios Enzimáticos/métodos , Escherichia coli/genética , Ensaios de Triagem em Larga Escala/métodos , Peroxidase do Rábano Silvestre/metabolismo , Hidroliases/genética , Engenharia de Proteínas/métodos , Sulfolobus solfataricus/genética
7.
Biochem Biophys Res Commun ; 523(3): 795-801, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31954521

RESUMO

The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNA-RNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From 102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1, which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity of compounds.


Assuntos
RNA Helicases DEAD-box/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , RNA Helicases DEAD-box/metabolismo , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos/métodos , Fator de Iniciação 4A em Eucariotos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Bibliotecas de Moléculas Pequenas/química
8.
Chem Commun (Camb) ; 56(11): 1629-1632, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-31939471

RESUMO

This article presents a new label-free fluorescence assay based on supramolecular self-assembly of cucurbit[7]uril and specific peptide Gly-Pro-Phe-Gly for monitoring DPP4 activity in clinical samples. It also displays a good potential application in high-throughput screening of DPP4 inhibitors.


Assuntos
Dipeptidil Peptidase 4/sangue , Ensaios Enzimáticos/métodos , Laranja de Acridina/química , Animais , Hidrocarbonetos Aromáticos com Pontes/química , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/química , Feminino , Corantes Fluorescentes/química , Humanos , Imidazóis/química , Limite de Detecção , Masculino , Camundongos Endogâmicos C57BL , Oligopeptídeos/química , Espectrometria de Fluorescência/métodos
9.
J Med Microbiol ; 69(2): 228-232, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31922949

RESUMO

Introduction. Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) from surveillance cultures is critical in supporting a good infection control programme. We implemented a new algorithm for CPE detection incorporating the NG Test CARBA 5 in January 2019.Aim. Our goals were to compare turnaround time (TAT), costs and staff requirements between the old and new algorithm, and to evaluate the performance of the CARBA 5 test directly on colonies grown on CARBA Smart agar.Methodology. We analysed and compared the TAT of CPE surveillance cultures processed using the old and new CPE screening algorithm. The total actual reagent costs and staff requirements for the new CPE algorithm were compared with the estimated costs and staff requirements of the old CPE algorithm.Results. Of 197 isolates included in the evaluation of the new algorithm, 64 were positive for carbapenemases by both CARBA 5 and Xpert Carba-R assay. Of the 133 that were negative, two were found to harbour NDM and IMI genotypes. Significant improvements in TAT were achieved with 88.7 % of cultures with CPE, reported on the same day as growth was observed on CARBA Smart agar compared to none in the old algorithm. The new algorithm incurred lower costs and, based on our workload, the new algorithm is estimated to save 28.9 man-hours annually.Conclusion. CARBA 5 performs well on colonies growing on CARBA Smart agar and significant improvements in TAT can be achieved without incurring additional costs or staff requirements.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas de Laboratório Clínico/métodos , Contagem de Colônia Microbiana/métodos , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/isolamento & purificação , Ensaios Enzimáticos/métodos , Algoritmos , Proteínas de Bactérias/genética , Técnicas de Laboratório Clínico/economia , Contagem de Colônia Microbiana/economia , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/diagnóstico , Ensaios Enzimáticos/economia , Humanos , Sensibilidade e Especificidade , beta-Lactamases/genética , beta-Lactamases/metabolismo
10.
Talanta ; 209: 120512, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892040

RESUMO

A label-free, ultra-sensitive and turn-on method for detecting RNase A has been developed using enhanced DNA-templated silver nanoclusters (DNA-AgNCs) as the fluorescence probe. In this system, an RNA strand, which can perfectly hybridize with DNA template of nanocluster synthesis, was applied to lock the fluorescent signal of DNA-AgNCs by forming an RNA/DNA duplex. Meanwhile, the hybridized RNA/DNA duplex was used as the substrate of RNase A. The fluorescence signal of AgNCs was restored due to the degradation of RNA by RNase A. From the fluorescence signal change of this system caused by RNase A, it was found that the fluorescence signal showed a positive linear relation with RNase A concentration in the range from 0.2 pg/µL to 10 pg/µL with a detection limit of 0.098 pg/µL. Except for potential inhibitor screening and the kinetic study of this enzyme, this strategy was further used for monitoring dynamic change of RNase A in living cells successfully. In summary, the simple and sensitive method for RNase A assay can be hopefully used for drug screening in vitro and in vivo.


Assuntos
DNA/química , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Microscopia de Fluorescência/métodos , Ribonuclease Pancreático/sangue , Animais , Sequência de Bases , Bovinos , DNA/genética , Células Hep G2 , Humanos , Cinética , Limite de Detecção , Hibridização de Ácido Nucleico , Ribonuclease Pancreático/química , Ribonuclease Pancreático/genética , Prata/química
11.
Biochemistry ; 59(7): 851-861, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-31951392

RESUMO

The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2, and E3 enzymes are together responsible for protein ubiquitination, thereby controlling cellular activities. Due to the numerous enzymes and processes involved, studies of ubiquitination activities have been challenging. We here report a novel Förster resonance energy transfer (FRET)-based assay for studying the in vitro kinetics of ubiquitination. FRET is established upon binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the free Ub sensor system (FUSS). Using Uba1, UbcH5, and CHIP as model E1, E2, and E3 enzymes, respectively, we demonstrate that ubiquitination results in decreasing FRET efficiency, from which reaction rates can be determined. Further treatment with USP21, a deubiquitinase, leads to increased FRET efficiency, confirming the reversibility of the assay. We subsequently use this assay to show that increasing the concentration of CHIP or UbcH5 but not Uba1 enhances ubiquitination rates and develop a novel machine learning approach to model ubiquitination. The overall ubiquitination activity is also increased upon incubation with tau, a substrate of CHIP. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3, or substrates and is thus likely compatible with any E1-E2-E3 combinations.


Assuntos
Endopeptidases/química , Ensaios Enzimáticos/métodos , Enzimas Ativadoras de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/química , Ubiquitinas/química , Animais , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Humanos , Cinética , Camundongos , Fragmentos de Peptídeos/química , Domínios Proteicos , Ubiquitina Tiolesterase/química , Ubiquitinação , Proteínas tau/química
12.
Yakugaku Zasshi ; 140(1): 81-90, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-31902889

RESUMO

We previously reported the association of the estimated glomerular filtration rate (eGFRcreat) calculated from the serum creatinine level (S-Cr) measured using the Jaffe method with the GFR (eGFRcys) estimated from the serum cystatin C level (CysC). However, few studies have compared the eGFRcreat using the enzymatic method with the eGFRcys. It is unclear whether there are differences in the results of renal function assessment. The purpose of this study was to compare the eGFRcreat calculated from the S-Cr with the eGFRcys calculated from the CysC in patients in whom the S-Cr and CysC were simultaneously measured using the enzymatic method, examine the correlations of respective parameters, and clarify physiological factors involved in differences among the parameters. The subjects were 1334 patients treated in 5 institutions. The mean values and correlation coefficient were statistically analyzed using Student's t-test and Pearson's test, respectively. Influential factors between formulae were analyzed using multiple regression analysis. The mean eGFRcreat was 67.0 mL/min/1.73 m2, being significantly higher than the mean eGFRcys (63.2). Multiple regression analysis showed that factors influencing differences in the S-Cr and CysC included the sex, age, serum albumin, and blood urea nitrogen BUN/S-Cr. Furthermore, factors involved in the overestimation of the eGFRcreat in comparison with the eGFRcys included the serum albumin and BUN/S-Cr. The differences between the eGFRcreat calculated from the S-Cr and eGFRcys were less marked than when adopting the Jaffe method in our previous study. However, the eGFRcreat were higher than the eGFRcys in patients with malnutrition or dehydration.


Assuntos
Creatinina/sangue , Cistatina C/sangue , Ensaios Enzimáticos/métodos , Testes de Função Renal/métodos , Insuficiência Renal Crônica/diagnóstico , Biomarcadores/sangue , Feminino , Taxa de Filtração Glomerular , Humanos , Masculino , Taxa de Depuração Metabólica
13.
J Chromatogr A ; 1609: 460438, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31447207

RESUMO

Plants are an important source of natural iridoids. This study demonstrates for the first time the acetylcholinesterase (AChE) inhibitory activity of iridoids belonging to the class of antirrhinosides. As iridoids distinguish the chemical composition of most species of the Plantaginaceae family, the active AChE inhibitors were investigated in the hydro-alcoholic extract of Anarrhinum pubescens Fresen. High-performance thin-layer chromatography (HPTLC) in combination with the AChE inhibition assay is a time and material saving methodology, and thus was employed to directly point to the individual enzyme inhibitors occurring in the plant. The effect-directed screening successfully discovered three active metabolites. These were characterized as antirrhinoside-derived iridoids. Two of these are here reported as newly isolated natural compounds. Identification of the two new metabolites was based on analysis of their collected spectroscopic data (HRMS, 1D and 2D NMR). Their structures were elucidated to be 6-O-, 6'-O-di-trans-cinnamoyl-antirrhinoside (1) and 5-O-, 6-O-difoliamenthoyl-antirrhinoside (3), while the previously known compound 6-O-foliamenthoyl-(6'-O-cinnamoyl)-antirrhinoside (2) was assigned by extensive analysis of its HRMS and HRMS/MS data. The activity of the isolated compounds was referred to the known AChE inhibitor rivastigmine, i.e. their activity were calculated and expressed as values equivalently to rivastigmine. This neuroprotective potential of iridoids mediated through AChE inhibition promote them to compete as natural curatives for neurodegenerative disorders like Alzheimer's disease.


Assuntos
Acetilcolinesterase/análise , Cromatografia em Camada Delgada/métodos , Ensaios Enzimáticos/métodos , Glucosídeos Iridoides/isolamento & purificação , Plantaginaceae/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Inibidores da Colinesterase/análise , Inibidores da Colinesterase/farmacologia , Fluorescência , Glucosídeos Iridoides/farmacologia , Metaboloma , Espectroscopia de Prótons por Ressonância Magnética
14.
J Chromatogr A ; 1609: 460454, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31443966

RESUMO

We propose a new capillary electrophoresis (CE)-based open-tubular immobilized enzyme microreactor (OT-IMER) and its application in acetylcholinesterase (AChE) assays. The IMER is fabricated at the capillary inlet (reactor length of ∼1 cm) with the inner surface modified by a micropore-structured layer (thickness of ∼220 nm, pore size of ∼15-20 nm). The use of IMER accomplishes the enzymatic reaction and separation/detection of the products in the same capillary within 3 min. The feasibility of the proposed method is evaluated via online analysis of the activity and inhibition of AChE enzymes. Such method exhibits good reproducibility with relative standard deviation (RSD) of less than 4% for 20 runs, and the enzyme remains over 82% of the initial activity after usage of 7 days. The IMERs are successfully applied to detect the organophosphorus pesticide, paraoxon, in three types of vegetable juice samples with a limit of detection of as low as 61 ng mL-1. Results show that the spiked samples are in the range of 89.6-105.9% with RSD less than 2.7%, thereby indicating its satisfactory level of accurate and reliable analysis of real samples by using the proposed method. Our study indicates that, with combination of advantages of both porous-layer capillary and CE OT-IMER, the proposed method is capable to enhance enzymatic reactions and to achieve rapid analysis with simple instrumentation and operation, thus would pave the way for extensive application of CE-based IMERs in a variety of bioanalysis.


Assuntos
Acetilcolinesterase/análise , Reatores Biológicos , Eletroforese Capilar/métodos , Ensaios Enzimáticos/métodos , Enzimas Imobilizadas/metabolismo , Inibidores da Colinesterase/análise , Sucos de Frutas e Vegetais , Cinética , Paraoxon/análise , Porosidade , Reprodutibilidade dos Testes , Espectroscopia de Infravermelho com Transformada de Fourier
15.
Artigo em Inglês | MEDLINE | ID: mdl-31676442

RESUMO

Pancreatic lipase (PNLIP) is a digestive enzyme that is a potential drug target for the treatment of obesity. A better understanding of its regulation mechanisms would facilitate the development of new therapeutics. Recent studies indicate that intestinal lipolysis by PNLIP is reduced by Angiopoietin-like protein 4 (ANGPTL4), whose N-terminal domain (nANGPTL4) is a known inactivator of lipoprotein lipase (LPL) in blood circulation and adipocytes. To elucidate the mechanism of PNLIP inhibition by ANGPTL4, we developed a novel approach, using isothermal titration calorimetry (ITC). The obtained results were compared with those of well-described inhibitors of PNLIP - ε-polylysine (EPL), (-)-epigallocatechin-3-gallate (EGCG) and tetrahydrolipstatin. We demonstrate that ITC allows to investigate PNLIP inhibition mechanisms in complex substrate emulsions and that the ITC-based assay is highly sensitive - the lowest concentration for quantification of PNLIP is 1.5 pM. Combining ITC with surface plasmon resonance and fluorescence measurements, we present evidence that ANGPTL4 is a lipid-binding protein that influences PNLIP activity through interactions with components of substrate emulsions (bile salts, phospholipids and triglycerides), and this promotes the aggregation of triglyceride emulsions similarly to the PNLIP inhibitors EPL and EGCG. In the absence of substrate emulsion, unlike in the case of LPL, ANGPTL4 did not induce the inactivation of PNLIP. Our data also prove that due to various interactions with components of substrate systems, the effect of a PNLIP inhibitor depends on whether its effect is measured in a complex substrate emulsion or in a simple substrate system.


Assuntos
Proteína 4 Semelhante a Angiopoietina/farmacologia , Fármacos Antiobesidade/farmacologia , Calorimetria , Ensaios Enzimáticos/métodos , Lipase/antagonistas & inibidores , Proteína 4 Semelhante a Angiopoietina/uso terapêutico , Fármacos Antiobesidade/uso terapêutico , Catequina/análogos & derivados , Catequina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Lipase/genética , Lipase/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Orlistate/farmacologia , Polilisina/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
J Chromatogr A ; 1611: 460577, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31591040

RESUMO

Ultraviolet radiation from sunlight causes DNA damage in skin cells by formation of photoproducts, mainly cyclobutane pyrimidine dimers (CPD), which are reverted by exogenous CPD-photolyase, preventing photoaging and skin cancer. High performance liquid chromatography tandem mass spectrometry method for quantification of CPD-photolyase activity was developed to search new enzymes sources for dermatology or clinical studies. The method was based in the enzymatic conversion of a 15mer oligonucleotide, containing a center cyclobutane thymidine dimer, to the restored 15mer oligonucleotide. Three ion pair reagent were evaluated by response surface methodology to increase mass intensities. Additionally, chromatographic separation of oligonucleotides was performed. The selected mobile phase was 15 mM diisopropylethylamine/20 mM hexafluoroisopropanol in methanol. The method allowed total separation between the oligonucleotides studied (resolution of 2.3) by using the core shell technology, which reduce the diffusion time of the analyte into the column, increasing the efficiency and minimizing the analysis time at 7 min. The mass spectrometry detection allowed a high selectivity and sensitivity. This is the first time where MRM modality has been employed with this specific purpose. Oligonucleotides recovery from reaction mixture was ∼ 94% and the limit of quantification was 13.4 nM for 15mer. The method was evaluated with a recombinant CPD-photolyase from Synechococcus leopoliensis using purified and crude protein extract. CPD-photolyase could be measured in terms of activity for enzymatic kinetics studies, for evaluation of UV-R effects in (micro)organisms and to identify new enzymes.


Assuntos
Proteínas de Bactérias/química , Cromatografia Líquida/métodos , Desoxirribodipirimidina Fotoliase/química , Ensaios Enzimáticos/métodos , Oligonucleotídeos/análise , Synechococcus/enzimologia , Espectrometria de Massas em Tandem/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Cinética , Dímeros de Pirimidina/química , Synechococcus/química , Synechococcus/genética
17.
Lett Appl Microbiol ; 70(1): 42-47, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31642085

RESUMO

The increasing frequency of class A KPC enzymes, class B metallo-ß-lactamases (MBLs) and class D OXA-48 enzymes in Enterobacteriaceae makes their early identification urgent. A simple commercial MASTDISCS combi Carba plus disc system (MAST-Carba plus) was designed for the identification of MBLs, KPC and OXA-48 carbapenemase genes in Enterobacteriaceae. To validate the MAST-Carba plus, a total of 77 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 84 isolates of noncarbapenemase-producing Enterobacteriaceae (non-CPE) were selected for differentiation of the genes of Enterobacteriaceae by MAST-Carba plus. Meanwhile, the carbapenemase genes such as blaKPC , blaIMP , blaVIM , blaNDM-1 and blaOXA-48 were detected by PCR (polymerase chain reaction). Thus, when considered on the basis of PCR results, the sensitivity of MAST-Carba plus detection of KPC strains is 82·3%, the specificity is 100·0%, the positive predictive value is 100·0% and the negative predictive value is 92·4%. For MBLs strains, the sensitivity is 100·0%, the specificity is 97·1%, the positive predictive value is 84·6% and the negative predictive value is 100·0%. For OXA-48 strains, the sensitivity is 100·0%, the specificity is 99·4%, the positive predictive value is 80·0% and the negative predictive value is 100·0%. Our findings suggest that MAST-Carba plus is a rapid and promising method for identifying the MBLs, KPC and OXA-48 carbapenemase genes in Enterobacteriaceae, which could be exploited in basic microbiology laboratory to prevent the transmission of CPE. SIGNIFICANCE AND IMPACT OF THE STUDY: Not only detection of carbapenemases but also identification of their genes accurately and rapidly in Enterobacteriaceae is still a major challenge for clinical laboratories in order to prevent the transmission of carbapenemase-producing Enterobacteriaceae (CPE). Therefore, this study aimed to evaluate the performance of a new rapid method (MASTDISCS combi Carba plus) for the identification of metallo-ß-lactamases (MBLs), KPC and OXA-48 carbapenemase genes in Enterobacteriaceae clinical isolates.


Assuntos
Proteínas de Bactérias/análise , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Ensaios Enzimáticos/métodos , beta-Lactamases/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , beta-Lactamases/genética , beta-Lactamases/metabolismo
18.
Biosci Biotechnol Biochem ; 84(1): 118-125, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31516066

RESUMO

We developed an enzymatic assay system enabling easy quantification of 4-aminobutyric acid (GABA). The reaction of GABA aminotransferase obtained from Streptomyces decoyicus NBRC 13977 was combined to those of the previously developed glutamate assay system using glutamate oxidase and peroxidase. The three-enzyme system allowing GABA-dependent dye formation due to the oxidative coupling between 4-aminoantipyrine and Trinder's reagent enabled accurate quantification of 0.2 - 150 mg/L GABA. A pretreatment mixture consisting of glutamate oxidase, ascorbate oxidase and catalase eliminating glutamate, ascorbate, and hydrogen peroxide, respectively, was also prepared to remove those inhibitory substances from samples. Thus, constructed assay kit was used to measure the GABA content in tomato samples. The results were almost the same as that obtained by the conventional method using liquid chromatography-tandem mass spectrometry. The kit will become a promising tool especially for the on-site measurement of GABA content in agricultural products.


Assuntos
4-Aminobutirato Transaminase/química , Aminoácido Oxirredutases/química , Colorimetria/métodos , Ensaios Enzimáticos/métodos , Peroxidase/química , Ácido gama-Aminobutírico/análise , Ampirona/química , Ascorbato Oxidase/química , Catalase/química , Cromatografia Líquida , Ensaios Enzimáticos/economia , Compostos Férricos/química , Ácido Glutâmico/química , Peróxido de Hidrogênio/química , Lycopersicon esculentum/química , Acoplamento Oxidativo , Proteínas Recombinantes , Streptomyces/enzimologia , Espectrometria de Massas em Tandem
19.
Biosens Bioelectron ; 148: 111834, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31706175

RESUMO

Efficient platforms for detecting telomerase activity are essential for early tumor monitoring and diagnosis. Herein, an enzyme-free electroanalytical strategy was developed for reliable and highly sensitive telomerase activity assay based on the increased electrochemical signals of methylene blue (MB) catalyzed by well monodisperse Au nanorods (AuNRs). In the presence of dNTPs and telomerase extracts, the assistant DNA 1 in the double stranded DNA can be extended to telomere repeat units (TTAGGG)n, which could form a hairpin structure by telomerase-triggered extension. The assistant DNA 2 was ingeniously dissociated from the double stranded DNA to combine with capture DNA. As a result, a large amount of AuNRs could be anchored on the surface of these sequences and used for electrocatalytic oxidation of MB. The developed biosensor showed a low limit of detection of 8.20 HeLa cells mL-1 and a wide dynamic range from 30 to 1.04 × 107 HeLa cells mL-1 for the determination of telomerase activity, which can provide a new way for telomerase activity assays in early diagnosis for cancers.


Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Nanotubos/química , Telomerase/análise , DNA/química , Técnicas Eletroquímicas/métodos , Ensaios Enzimáticos/métodos , Células HeLa , Humanos , Nanotubos/ultraestrutura
20.
Methods Mol Biol ; 2041: 197-207, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646490

RESUMO

Adenosine 5-triphosphate (ATP) functions in the central nervous system as an extracellular signaling molecule. While much progress has been made in understanding the circumstances under which it is released, from in vitro preparations, in vivo has proven more challenging. Microdialysis followed by high-performance liquid chromatography has been employed to demonstrate a spike in extracellular concentrations under some pathological conditions including seizures, but this method lacks the sensitivity to detect extracellular ATP at concentrations present under normal physiological conditions. An alternative approach, the use of amperometric, enzyme-based microelectrode biosensors for measuring extracellular ATP in vivo have been employed in the rabbit. Here, we describe a protocol for measuring ATP concentrations using these biosensors in the mouse, simultaneously with electroencephalogram recordings. This approach is ideal for investigating the relationship between ATP release and seizures.


Assuntos
Trifosfato de Adenosina/análise , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Ensaios Enzimáticos/métodos , Microeletrodos , Convulsões/metabolismo , Animais , Camundongos , Convulsões/patologia
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